The overall objective of this application is to rapidly develop much needed alternative plague vaccine candidate formulations based on our recent discovery that a modified Y. pestis V antigen (LcrV) based DNA vaccine was highly effective in protecting experimental mice against lethal intranasal challenge with Y. pestis. Our preliminary data suggested that good protection resulted from improved antigen presentation and possibly through improved Th1 type immune responses. This is the first time a DNA-based plague vaccine induced complete protection against lethal mucosal challenge in any animal model. We now propose to conduct vigorous validation and testing in the first 18-24 months to develop a subunit plague vaccine formulation using this novel modified V antigen as the core protective component. The protection efficacy of this subunit vaccine, delivered in the forms of DNA, protein, or a combination of both, will be evaluated against lethal Y. pestis challenge via the airway mucosa. Additional immunological studies will be conducted to confirm the improved efficacy of this modified V antigen. We also propose to continue our search for additional protective Y. pestis antigens to be incorporated into the above primary formulation so that a true multi-gene plague vaccine can be developed to achieve broader protection to discourage attempts to engineer vaccine resistant Y. pestis strains. This search will focus primarily on antigens involved in the function of the Y. pestis Type III secretion apparatus including YopB, YopD and YscF. A variety of combination formulations will be tested among these candidates and the modified V antigen to identify the formulation that can provide the most effective protection. This project will employ the most advanced DNA immunization technology in combination with small-scale protein production to quickly develop potential subunit-based plague vaccine products.